Fig. 1: Overview of the paper-based multiplexed vertical flow assay (xVFA) and point-of-care diagnosis of Lyme disease (LD).

a Transmission of Borrelia burgdorferi through the bite of Ixodes ticks and the presentation of various antigens generating an immune response from the host. b Comparison of incidence of LD cases in the northeastern US from 2000 and 2019 indicating an increase in the incidence of cases due to the growing population of ticks. Worldwide incidence of LD in 2019. Legend indicates the number of cases per 100,000 people. c Centralized laboratory-based two-tier serology of LD uses relatively expensive instruments and trained personnel, resulting in high turnaround time and cost per test. d Point-of-care xVFA assay using low-cost paper layers and a smartphone reader that provides results for a multiplexed LD assay in <20 min. e The xVFA contains a selected peptide panel immobilized on a nitrocellulose membrane that reacts with IgM and IgG antibodies from LD patient serum. f Stability of modVlsE-FlaB peptide and VlsE recombinant protein indicating a loss in performance of a protein immobilized assay by more than 50% over a 90-day period. Data were presented as mean values ± SD, with standard deviation indicating three replicates (N = 3). g Combining IgM and IgG detection in a single xVFA assay enhances the sensitivity of an individual immunoreaction spot (N = 3). Data were presented as mean values ± SD. h Smartphone-based portable reader and automated image processing of the signals from the peptide panel before and after the assay, yielding normalized signal intensities. The individual peptide spots are analyzed using a multiplexed model to classify samples as either LD positive or negative. Panels a, c, d were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en).